Benzyl alcohol extraction of antipernicious anemia concentrate



Patented Oct. 6, 1953 BENZYL ALCOHOL EXTRACTION OF ANTI- PERNICIOUSANEMIA GONCENTRATE Frank R. Koniuszy, Rahway,

and Norman G.

Brink and Karl Folkers, Plainfield, N. .L, as-

signors to Merck & 00., Inc.,

Rahway, N. J., a

corporation of New Jersey No Drawing. Original application November 5,

1948, Serial N 0.

58,596. Divided and this application June 22, 1951, Serial No. 233,116The portion of the term of the patent subsequent to August 19, 1969, has

been disclaimed Claims. (01. 167-74.6)

This invention is concerned with a new and improved process forpurifying the anti-permcious anemia concentrate which is obtained fromcrude commercial liver preparations or other biological sources.

This application is a division of our application Serial No. 58,596,filed November 5, 1948, now Patent No. 2,594,314.

More particularly, our invention relates to a new and improved solventextraction process whereby it is possible to treat a crude commercialliver preparation, or a crude preparation obtained from other biologicalsources, which commercial preparation is of relatively low potencyagainst pernicious anemia, in order to free it from interferingimpurities, thereby securing a much improved concentrate of relativelyhigh potency, which new product is very valuable for use in anemiatherapy. Our invention is also concerned with the new concentrates ofhigh potency thereby produced.

The liver concentrates, and other preparations intended for treatingpersons afllicted with pernicious anemia, which are availablecommercially at the present time are all of relatively very low potency.Moreover, they often contain interfering materials which, in allinstances to a large extent, and in some cases completely, interferewith the potential effectiveness of these preparations when employed inthe treatment of pernicious anemia. It has therefore been of the utmosttherapeutic importance to prepare concentrates of the active principles,or active factors, present in crude commercial concentrates, so that aproduct of high effectiveness for use in anemia therapy will be secured.

This invention is concerned with a new and improved process involvingthe solvent extraction of commercial concentrates and it is possible, bypurifying these commercial concentrates in accordance with our process,to obtain a purified material of greatly increased potency, ranging froma minimum of a four-fold increase in potency, to as high as a TOO-foldincrease in potency. These surprising increases in potency of theproducts when employed in anti-pernicious anemia therapy are securedeven When the starting material is a concentrate of very low potency,for example one having a potency of only 1000 units per milligram orless.

It is accordingly the principal object of our invention to provide a newand improved method for purifying anti-pernicious anemia concentrates,obtained from crude commercial liver preparations and other biologicalsources, which method will permit the obtainment of a highly purifiedproduct, having a potency increased to a remarkable degree as comparedwith the potencies of commercial crude concentrates.

It is another object of this invention to render available a process forthe purification of the commercially available liver preparations, aswell as preparations obtained from other biological sources which areuseful in the treatment of antipernicious anemia, which process willinvolve solvent extraction of the commercial concentrates o-rpreparations, and which will result in an improved product of very highpotency. This product of high potency is secured even when thecommercial preparation treated has relatively very low potency when usedfor treating pernicious anemia.

It is still another object of our invention to utilize in the solventextraction process, which permits the purification and obtainment ofhighly concentrated products, extraction solvents such as the alkylphenols. Previous to the development of the process herein disclosed, itwas believed that the active principles or factors present in liverconcentrates, and in other concentrates eiTective against perniciousanemia which are obtained from other biological sources, wereproteinaceous or polypeptide in their nature. It was also Well knownthat proteinaceous substances generally do not lend themselves readilyto methods of purification involving extraction with organic solvents.It was therefore assumed that the active principles in theseconcentrates could not be efiectively extracted by treatment withorganic solvents. Although, in previous methods of purification, phenolhad been utilized to extract the anti-pernicious anemia factor fromsolid concentrates such as crude residues or charcoal adsorbates, it wasgenerally considered impracticable (if not impossible), to utilizephenol in a two-liquid phase extraction system. One

reason for this is the high solubility of phenol in water. Moreover, itis very diflicult to remove phenol completely from an aqueous solutionby simple extraction with immiscible organic sol- M vents.

In spite of the widespread belief that a solvent extraction processcould not be utilized in the purification of crude commercial liverconcentrates, it has now been discovered that a solvent extractionprocess, particularly a solvent extraction process employing the alkylphenols, is particularly effective in producing purified concentrates ofa surprisingly high potency.

We have found it desirable to utilize as starting materials in thepreparation of our new and improved anti-pernicious anemia concentratesby solvent extraction methods a commercial crude liver extractconcentrate such as, for example, Dakin and Wests liver fraction, orAnahaemin. This product, and the method by which it is prepared, isdescribed by Dakin and West in an article in the Journal of BiologicalChemistry, 1935, volume 109,, page 489. Clinical notes on its use in thetreatment of pernicious anemia will be found in the articles of Ungleyet al., and of Wilkinson, in the British publication Lancet for February'15, 1936. These articles are in volume 230 and begin, respectively,

at pages 349 and 354. While Anahaem-in is :a

preferred crude commercial liver preparation, useful as a startingmaterial in carrying out our solvent purification process, we are notrestricted to utilizing this crude material as the starting material,and may use various other anti-perni'cious anemia concentrates obtainedfrom crude commercial liver preparations -or other biological sources.Illustrations of such suitable starting materials are given in theexamples, given below, of our invention.

In carrying out our invention, in accordance with our preferredprocedure, we first purify the crude liver concentrate bychromatography. The resulting product :is then dissolved in water, thesolution acidified and extracted with an alkyl phenol, such as amylphenol. This .alkyl phenol extract is thendiluted'with petroleum etherand extracted with water. The resulting water extract is washed withmesityl oxide, the residual mesityl oxide removed by washing withchloroform, and the purified water solution then extracted with benzylalcohol. The benzylalcohol extract is then diluted with chloroform .andextracted with water. Residual benzyl alcohol is removed from thisaqueous extract by extraction with chloroform. From the purified waterextract there is recovered -a:so1id concentrate of theanti-perniciousanemia factor of surprisingly high potency.

While in our preferred process we utilize all of the individual solventextraction steps specified, it is by no means necessary to utilize allof these extractionsteps, and we have obtained products of greatlyenhanced therapeutic activity by utilizing one or more of the individualsteps. Thus, we may .utilize an extraction procedure wherein the aqueoussolution of the anti-pernicious anemia concentrate obtained from crudecommercial liver preparations or other sources is 'extracted with animmiscible organic solvent phase containing an 'alkyl phenol, such asamylphenol, or with benzyl alcohol. Or, if desired, some of theimpurities present in the active solution may be removed by washing theactive solution with mesityl oxide or with 'fiuorobenzene. By means ofany of these individual steps it is possible to prepare a product ofenhanced therapeutic activity, starting with a relatively impurecommercial liver preparation. However, as previously stated, ourimproved solvent extraction procedure involves the utilization of all ofthe steps referred a to above and illustrated in the diagram givenbelow.

Our complete or preferred process may be described more fully asfollows. A crude commercial liver extract, or other concentrate of thepernicious .anemia factor, is ifirst dissolved in water. Often itadvantageous to carry out a preliminary purification of this watersolution by passing the solution through a chromatographic columncontaining alumina, as described in the copentl'ing application ofFolkers and Shavel, Serial No. 20,106, filed April 9, 1948, now PatentNo. 257-35702. However, in accordance with our preferred process, thischromatographic purification step does n'ot'r'equire careful fractionalelution, as simple passage of the solution through the column, followed:by washing with a portion of the .same solvent, is sufficient to effectthe rough purification found to be desirable as a preliminary step.

The -eluate is then concentrated to dryness, and the resulting residuedissolved in water. The

' solution is then acidified by the addition of hydrochloric acid, to apH of approximately 1.0, whereupon it is extracted several times with analk-yl phenolsuch as am-yl phenol. The combined amyl phenol solution isthen washed several times with 5 "aqueous sodium bicarbonate solutionand with water. It is then diluted with petroleum ether and extractedseveral times with I water. The combined waterextracts are washed withseveral portions of mesityl oxide. Further washing with chloroformremoves the residual mesityl oxide i that may have remained :in theaqueous extract.

This aqueous solution is then extracted with several portions of benzylalcohol. The combined benzyl alcohol extracts are diluted with -chloro.form and extracted with several portions of water.

The residual .benzylalcohol is next removedfrom the water extract bywashing the extract with chloroform. When the extract is then subjectedto freeze drying, .utilizingany commercially available .method, such asrreezedrying .by theLyophil- Cryochem process, there results .apink-yellow solid which is highly effective for the treatment ofpernicious anemia.

Gur improved process maybe illustrated :by the v followingdiagrammatical representation:

Crude liver concentrate activity 1,000 u.-/mg.

Dissolvedin watcnacidified to pH 1.0

Discard extracted l aqueoussolution Extraction with alkyl phenol TSlJ'Ghas amylph'enol Wash alkylrphenol solution with aqueoussodiumbicarbonateDilution of alkyl plienohextract with petroleum ether Discard extractedalkyl 1 7 ,phenol solution Extraction with-water Wash with mesityl oxideWash with chloroform to remove residual mesityl oxide Aqueous solutionextracted with benzyl alcohol Dilute with chloroform and extract withwater Removal of residual benzyl alcohol by extraction with chloroformas illustrative and not as restrictive, since various changes could bemade therein without departing from the scope of our invention asdefined in the appended claims.

Example 1 A chromatographic column 2 x 10 cm. was prepared using a waterslurry of 14.5 grams of alumina. To the column a solution of 1.3 gramscrude commercial liver concentrate, Anahaemin, in 10 cc. water wasadded. This initial material had an activity of about 700 units/mg. Thecolumn was eluted with water and the first two fractions totalling 50cc. were concentrated to dryness in the frozen state in vacuo to yield540 mg. of a white solid.

This was dissolved in 25 ml. of water and extracted twice with 15 ml.portions of amylphenol. (The resulting emulsions were centrifuged toseparate the layers.) The clear amylphenol extract was diluted with 150ml. of petroleum ether, and this solution was extracted twice with 25ml. portions of water. After washing the aqueous extract twice with 25ml. portions of chloroform, it was concentrated to dryness in the frozenstate in vacuo to yield 169 mg. of a cream colored solid. This creamcolored solid had an activity range of 1,850 to 4,000 units per mg, andwhen 15 mg. of it was given to a pernicious anemia patient it producedan excellent characteristic reticulocyte response in 5 days andsubsequent restoration of the number of mature erythrocytes to a normalvalue.

Example 2 Five hundred forty-three mg. of the antipernicious anemiafactor concentrate purified by chromatograph over alumina, as describedin Example 1 (assay about 4800 u./mg.), was dissolved in 25 ml. ofwater. This solution was extracted four times with 20 m1. portions ofbenzyl 6'. alcohol (the resulting emulsions were centrifuged to separatethe layers). The aqueous layer was washed twice with 25 ml. portions ofchloroform and the chloroform washings were added to the benzyl alcoholextract. The combined benzyl alcohol extract was diluted with ml. ofchloroform and then extracted four times with 25 ml. portions of water.The aqueous extract was washed with 25 ml. of chloroform, frozen andconcentrated to dryness in vacuo to yield 70 mg. of a pale yellow solidthat showed a Lactobacillus lactic Dorner growth activity of about30,000 units per mg. 1

Example 3 450 mg. of the amylphenol soluble fraction of the liverconcentrate with an LLD activity range of 1,850 to 4,000 u./mg.(Example 1) was dissolved in 20 ml. of water. The solution was acidifiedto pH 1.0 with hydrochloric acid and extracted three times with 10 m1.portions of amylphenol. The combined amylphenol extract was Washed with15 ml. of 5% aqueous sodium bircarbonate solution and with 10 ml. ofwater. The amylphenol solution was diluted with 1,000 ml. of petroleumether and this solution was extracted four times with 50 ml. portions ofwater. The aqueous extract was washed twice with 25 ml. portions ofchloroform and twice with 25 ml. portions of vmesityl oxide. The aqueoussolution was then frozen and concentrated to dryness in vacuo to yield202 mg. of a white solid possessing an LLD activity range of 3,500u./mg. to 8,500 u./mg. This material was active clinically at 3 mg.

Example 4 101 mg. of the product of Example 3 was dissolved in 5 ml. ofwater. The resulting solution was washed ten times with 5 ml. portionsof redistilled mesityl oxide and four times with 5 ml. of redistilledfiuorobenzene. The aqueous solution was lyophilized. 83 mg. of a whiteproduct was obtained which had an activity range of 10,000 to 15,000u./mg. and which gave an excellent clinical response when given to apernicious anemia patient in a 2 mg. dose.

Example 5 42 mg. of the product of Example 4 in 2 ml. of water wasextracted four times with 2 ml. of benzyl alcohol. The benzyl alcoholextract was diluted with 400 ml. of chloroform which was then extractedfour times with 20 ml. of water. After washing the aqueous extract with10 ml. of chloroform it was lyophilized to yield 14.4 mg. of a solidpossessing an activity range of 27,000 to 66,000 u./mg.

Example 6 2 grams of the anti-pernicious anemia concentrate obtainedaccording to the procedure shown in Example 5 (benzyl alcohol solublefraction with an activity range of 66,000 to 113,000 u./mg.) wasdissolved in 25 ml. of water, and the solution was acidified to pH 1.0with hydrochloric acid. The solution was extracted six times with 25 ml.portions of amylphenol. Emulsions were broken by centrifuging, ifnecessary. The combined amylphenol extract was washed twice with 25 ml.portions of 5% aqueous sodium bicarbonate solution, and once with 25 ml.of water. The amylphenol solution was now diluted with 1500 ml. ofpetroleum ether and extracted six times with 100 ml. portions of water.The

combined aqueous extract was washed three ml. portions ot'mesityloxid'e'ar-id clarified withtwo 25: ml. washes oi chloroform.Lyopnili'zation of the aqueous extract yielded 426 mg. of a pink-yellowcolored solid possessing an; activity range of 303,000 to 575,000'1L/mg.

Example 7 gm. of crude commercial. liver concentrate (with apotency of4,000 to: 9,000 u-./mg..) was dissolved in 100: ml. of water. Thesolution was added. to the top of. a chromatograph column packed with.300 grams of acid-washed alumina. The column was washed with water, andthe first 800 ml. of solution passing through the column was collectedand acidified to pH 1.0 with hydrochloric acid. The solution wasextracted with. six: 100 ml. portions or amylphenol, and the combinedamyl'ph'enolextract was washed. withtwo: 1160;.11112'130121310115 of 5%sodium bicarbonate s'oltrtion, witl'r 100 ml. of water, andthen dilutedwith. 2 liters of petroleum ether. The diluted solution was extractedwith six 150' ml. portions or water and the combined aqueous extract waswashed three times with 100 ml. portions of redistilled mes-ityloxideand twice 100 ml. portions of chlorofiorm. The clarified aqueoussolu tion was now extracted six times with 100 ml. portionsof benzylalcohol. The combinedbenzyl alcohol extract was diluted with 2 liters ofchloroformand then extracted with six. 150 ml. portionsof water; Theaqueous extract was clarified by washmg; with two100'ml. portions ofchloroform. Lyophilizati'orr yielded 304 mg. of a yellowish-pinkcolored: solid, possessin an activity range of 700,000 to 830,000 unitsper mg. Example- 8 One grant of a dried concentrate was secured bycultivating a strain of the micro-organism strepwmyces g-ris'eus on asuitable nutrient medium and recovering the active material from themature brothfollowing the procedure disclosed in the application ofFrederick A. Kuehl' and Louis Chalet, Serial No. 18,848, filed April 3,

194a", new Patent no. 2,505,053. This dried con centrate had an activityof about 4300' u./mg. It was dissolved in- 25 ml. of water. The solutionwas acidified to pH 2: with hydrochloric acid and extracted once. with25 ml. of amylphenol and then three times with 15' ml. portions. ofamylphenol; The combined amylphcnol extracts were washed twice with '75m1. portions of 5% aqueous sodium bicarbonate, diluted with petroleumether and extracted with seven. 50 ml. portions of water. Lyophflizationgave 180 mg; or product possessing about. 20,000 u./mg.. activity; I

The highly potent concentrates, secured by following the proceduredescribed in Examples 1, 3 and 4, and having the properties therein.-described, were tested clinically on patients having pernicious anemia.All gave excellent responses; Moreover, the improvements resulted fromthe administration of very small doses of the new concentrates of highpotency. Thus, doses ranging. from 2- mg. to: 15 mg. per patient,-depending oh. the purity of the individual. samples and the severity ofthe patients condition, were found tobeefiective. These low dosages aretobe compared with the dosages necessary with the commercially-availableconcentrates, such. as, Anahaemin, of which doses ranging from 100 mg.to 200- mg.- per patient have been reported 8 in the literature asrequired in order to produce good clinical responses.

The assays given in the above examples are on the basis of 1,000 unitsfor a standard. material, and are determined as microbiological growthresponse assay. In most or the experiments listed in the examples of ourimproved process given, the assay values" have been reported as ranges,due to variations in the assay.

It should be understood that various changes and modifications can bemade in our invention as herein described Without departing from thescope thereof. Accordingly, such changes and modifications as are withinthe purview of the appended claims are to be regarded as part of ourinvention.

We claim:

1. The process for preparing a concentrate having enhanced activity fortreating pernicious anemia and for promoting the growth of LactobacillwsLactis Dorner from a crude concentrate of biological origin containingsaid active material, which comprises subjecting an aqueous solution ofthe crude material containing the active material to extraction withbenzyl alcohol, and recovering from the resulting benzyl alcohol extracta purified concentrate of enhanced activity.

2. The rocess as defined in claim 1 wherein the starting concentrate isa crude liver preparation.

3. The process as defined in claim 1 wherein the starting concentrate isa crude material obtained from Streptomyce's griseus fermentati'on.broth.

4. The process for preparing a concentrate having enhanced activity fortreating pernicious anemia and for promoting the growth of LactobacillusZactis Dorner from a crude concentrate of biological. origin. containingsaid activev material, which. comprises subjecting an aqueous solution.of the crude material. containing the active material to extraction withbenzyl alco-' hol, diluting the resulting benzyl alcohol extract with. amiscible water insoluble solvent, extract-- ing the resulting. dilutedbenzyl alcohol extract with. water, and. recovering from theaqueousextract. apuri-fiedconcentrate of enhanced; activity.

. 5. The process for preparing a concentrate having enhanced activitytor treating perniciousanemia and for promoting the growth ofLa'ctobacillus lactis-Dorner from a crude concentrate of bio-logicalorigin. containing said active mate-- rial, which. comprises subjectingan aqueous solution of the crude material containing the active-materialto extraction with. benzyl alcohol; adding chloroform to the resultingbenzyl alcohol extract,v extractin the resulting benzyl alcohol chloroforrm solution with. water, and recovering from-the: aqueous extracta purified con-- centrateot enhanced activity.

. R. KONIUSZY.

NORMAN G. FOLIQ1R3.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 2,134,256 Leland Oct. 25, 1938 2,607,717" Brink s- Aug. 19.,1952.

1. THE PROCESS FOR PREPARING A CONCENTRATE HAVING ENHANCED ACTIVITY FORTREATING PERNICIOUS ANEMIA AND FOR PROMOTING THE GROWTH OF LACTOBACILLUSLACTIS DORNER FROM A CRUDE CONCENTRATE OF BIOLOGICAL ORIGIN CONTAININGSAID ACTIVE MATERIAL, WHICH COMPRISES SUBJECTING AN AQUEOUS SOLUTION OFTHE CRUDE MATERIAL CONTAINING THE ACTIVE MATERIAL TO EXTRACTION WITHBENZYL ALCOHOL, AND RECOVERING FROM THE RESULTING BENZYL ALCOHOL EXTRACTA PURIFIED CONCENTRATE OF ENHANCED ACTIVITY.